This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method
was developed for the detection and differentiation of wild-type and vaccine strains
of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding
to the highly conserved region of the CDV genome were used as a common primer pair
in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains,
were used as the forward primer together with the common reverse primer P4 in the
second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine
strain, were used as the forward primer together with the common reverse primer P4+P6
in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine
strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in
the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed
samples of vaccine and wild-type strains. No amplification was achieved for uninfected
cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV),
canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR
method was used to detect 30 field samples suspected of canine distemper from Heilongjiang
and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples,
were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method
can be used to effectively detect and differentiate wild-type CDV-infected dogs from
dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological
surveillance.

Introduction

Canine distemper (CD) is a highly contagious and fatal disease of dogs caused by the
canine distemper virus (CDV), which is a single-stranded negative RNA virus belonging
to the Morbillivirus genus within the Paramyxoviridae family. Other members of the genus include measles virus (MV) and rinderpest virus
(RPV). The genome of CDV is approximately 15,690 nucleotides (nt) in length, containing
several genes encoding N, P, M, F, H, and L proteins. Only one serotype has been characterized
[1].

A large number of dogs, minks, foxes die from CDV infections every year, causing significant
economic losses[2]. Previous studies[3,4] have reported that vaccinated dogs were infected with CDV in Europe and Japan. Harder
et al. also reported that there are marked differences between wild-type and vaccine
strains of CDV[5], thus whether CDV vaccine strains are able to provide protection from the current
strains of CDV remains a question. It is difficult and necessary to discriminate between
wild-type and vaccine strains because the attenuated CDV vaccine is used widely in
China. So a method to specifically detect the wild-type CDV strains is necessary.
The multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method
could be used to effectively detect and differentiate between wild-type CDV-infected
dogs from dogs which were vaccinated with CDV vaccine, which would make it useful
in clinical diagnosis and epidemiological monitoring.

Study Design

With the help of Oligo6 software, six primers were designed based on the genomic sequences
of CDV strains published in GenBank (CDV strains: Onderstepoort, Convac, A75/17, Rorkborn,
Snyder Hill, Lederle, et al). Forward primers P1 (5'-AAATCCTGTGTTACCCGCTC-3'), P2
(5'-TGGTGGCTCTGCAATATGAA-3'), and P3 (5'-AATGAATGGATGCCTGGGGTTT-3') were used as the
primer specific for CDV species, wild-type strains, and vaccine strain Onderstepoort,
respectively. Primer P4 (5'-ACGTCCTGGACCCTAAGTTTTG-3') was used as a shared reverse
primer. P5(5'-GGTTTTATAAAAGATT), p6(5'-ATCTAGAGGTAA-3') were used as the primer specific
for different CDV vaccine strains. Primer pairs P1/P4, P2/P4, P3/P4 and P5/P6 were
expected to generate a product of 600, 247, 177 bp and 177 bp, respectively. CDV vaccine
(CDV-A strain) and wild-type strains are maintained in the Harbin Veterinary Research
Institute. A total of 30 field samples (lung, spleen, liver, or bladder) were collected
from different farms in Heilongjiang and Jilin provinces of China.

Multiplex reverse transcription-nested polymerase chain reaction

Total RNA was extracted from the infected cells with TRIzol® reagent in accordance with the manufacturer's instructions. cDNA synthesis reaction
was performed by polymerase chain reaction as described elsewhere using primer Moloney
murine leukemia virus reverse transcriptase (M-MLV RT)[6-10]. The first-round PCR was performed with primers P1 and P4. A nested PCR was performed
in a total volume of 25 μL containing the first-round PCR products diluted tenfold
as well as each of primers P2/P4, P3/P4, or P2, P3/P4[10-12]. The RT-nPCR products were visualized by electrophoresis in a 2% (w/v) agarose gel.

30 samples in Heilongjiang and Jilin provinces, and Fifty-one field samples from dogs,
raccoons, foxes, and minks in Shandong provinces were assayed by RT-nPCR; the background
of the 51 field samples is listed in table 1. All the positive field samples wild-type strain were confirmed by Rapid test which
BioNote, Inc. produced.

Phylogenetic analysis and detection of CDV in field samples by RT-nPCR

Two of the field samples from Heilongjiang province were selected for amplification
of the H gene of CDV by RT-PCR with primers P5 (5'-CCAATTCATCCAAGCTGTCC-3') and P6
(5'-GGGATTTGAACGGTTACATGAG-3'). The amplified products were cloned and sequenced,
and the sequences were aligned with the H genes of a number of CDV strains available
in GenBank using the MegAlign function of the DNAStar software package. Thirty field
samples from dogs, foxes, and raccoons in Heilongjiang and Jilin provinces were also
assayed by RT-nPCR.

Results

Determination of the application conditions of the multiplex RT-nPCR

After the application of the first-round PCR, primers P2 and P4, and P3, P4, P5 and
P6 together, were used to amplify the vaccine and wild-type strains, respectively,
at different anneal temperatures. According to the result, when the anneal temperature
was from 49-54°C, there was only one specific band. Primers P2, P3, P4, P5 and P6
were used to perform RT-nPCR with different anneal temperatures. Only one specific
band was observed at an anneal temperature from 49.5-54.5°C, with the most distinct
band appearing at 51.5°C. Thus, 51.5°C was chosen for the RT-nPCR.

Specificity of the multiplex RT-nPCR

A fragment of 247 and 177 bp was amplified from CDV wild-type strain and the vaccine
strain, respectively. Two bands of 247 and 177 bp were detected simultaneously from
the mixed genomic RNA of the CDV wild-type and vaccine strains. Amplification was
not possible for non-CDV viruses, such as CPV, CAV, CCV, RV, NDV-infected cells, and
uninfected cells by RT-nPCR (Fig. 1).

Sensitivity, applicability, and repeatability of the multiplex RT-nPCR

Serial 10-fold dilutions of CDV vaccine strain were subjected to amplification by
multiplex RT-nPCR. The lowest limit of detection with this method was shown to be
0.1 TCID50. Of the 30 field samples, 20 tested positive for CDV, among which, 15 showed presence
of wild-type viruses, and 5 showed presence of vaccine strain. Three independent inter-
and intra-assay replicates of the multiplex RT-nPCR gave consistent results, indicating
the repeatability of the assay.

Phylogenetic analysis based on H gene

A phylogenetic tree based on the H genes of various CDV strains was generated using
the MegAlign of DNAStar software. As shown in Fig. 2, the selected two samples were grouped into wild-type viruses and belonged to a genotype
that is obviously different from the CDV vaccine strain.

Discussion

To date, CDV remains one of the most important canine diseases worldwide. Surveillance
represents a primary concern in the control of CDV. In addition to traditional methods
using virus isolation, several promising antigen-ELISA, AGP, and FA methods have been
developed and evaluated. However, these methods are laborious and time consuming.
Moreover, these tests cannot differentiate CDV wild-type strains from vaccine strains[13,14].

With the recent availability of genomic sequences, molecular diagnostic methods for
detection of viruses have significantly improved[15]. Complementary DNA and RNA probes have been used to detect RNA and mRNA of the CDV
genome with improved specificity and sensitivity[6,7]. Primers P1 and P4 (specific for CDV and conserved among CDV species), primer P2
(specific for CDV wild-type strain), and primer P3, P5 (specific for CDV vaccine strain),
were selected from the well-conserved regions of the gene encoding matrix protein.
A fragment of 600 bp was consistently amplified by RT-PCR with P1/P4 for either CDV
vaccine strain or wild-type strain. The nested PCR with P2/P4 generated a 247 bp fragment
only for the wild-type strain, while P3/P4, P5/P6 generated a same 177 bp fragment
only for the vaccine strain, and both fragments could be amplified from the mixture
of CDV vaccine and wild-type strains. No amplification was obtained for NDV and other
common canine viruses, such as CCV, CPV, CAV, RV, and uninfected cells control, indicating
the high specificity of the method. The method was sensitive, in that it could detect
as little as 0.1 TCID50 of the virus. The selected two samples were classified into a branch which belongs
to the wild-type strain, but constituting a genotype different from that of CDV vaccine
strains, as revealed by phylogenetic analysis based on the sequences of the H gene
region, which is believed to be the most reliable classification and genetic typing.
RT-nPCR was used to detect the 30 field samples in Heilongjiang and Jilin provinces;
20 of the field samples were CDV-positive, among which 15 were wild-type strain, and
5 were vaccine strain.

Caideron et al [16] performed an extensive phylogenetic and molecular evolution analysis on complete
sequences of all CDV genes to assess the role of selection and recombination in shaping
viral genetic diversity and driving the emergence of CDV in non-dog hosts. They tested
the specific hypothesis that molecular adaptation at known receptor-binding sites
of the haemagglutinin gene is associated with independent instances of the spread
of CDV to novel non-dog hosts in the wild. The selected two samples isolated from
dogs were classified into a branch which belongs to the wild-type strain, but constituting
a genotype different from that of CDV vaccine strains, as revealed by phylogenetic
analysis based on the sequences of the H gene region, which is believed to be the
most reliable classification and genetic typing. RT-nPCR was used to detect the 51
field samples in Shandong provinces; 36 of the field samples were CDV-positive, among
which 20 were wild-type strain, 16 were vaccine strain, and 4 were co-infected by
wild-type and vaccine strains. In summary, the multiplex RT-nPCR developed in this
study is a highly specific and sensitive assay for the rapid detection and differentiation
of wild-type and vaccine strains of CDV.

For lack of other vaccine strains except CDV Onderstepoort strain, the primers P5
and P6 were designed, but could not use in this time, however, much work needs to
be done before a conclusion that this method has an outstanding performance with other
CDV vaccine strains. In summary, the multiplex RT-nPCR developed in this study is
a highly specific and sensitive assay for the rapid detection and differentiation
of wild-type and vaccine strains of CDV.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

WS, SZ, ZW carried out the experiments and wrote the manuscript. SC conceived the
studies and participated in experimental design and coordination. All authors read
and approved the final manuscript.

Acknowledgements

The study was partly supported by funds from the Chinese state National Hightech
R&D Program (863 Program-2007AA100606). We would like to thank Dr Huaji Chou's help
to revise the paper.